Technical Abstract:
The endoplasmic reticulum (ER) is a dynamic network that consists of numerous regions or subdomains with discrete morphological features and functional properties, including those involved in protein and oil-body formation, anterograde transport of secretory proteins, the exchange of macromolecules with plastids and mitochondria, and lipid biosynthesis. While it is generally accepted that these subdomains differ in their protein and perhaps lipid compositions, a clear understanding of how they are established and maintained or how they are related to each other in terms of the overall organization of the ER has not been well established. Here we show that two tung (Vernicia fordii) membrane-bound glycerol-3-phosphate acyltransferase (GPAT) enzymes, GPAT8 and a newly-identified putative GPAT with homology to mammalian GPAT3, GPAT9, localize in tobacco BY-2 cells to the same ER subdomain that contains tung type 2 diacylglycerol acyltransferase (DGAT2), but is distinct from the ER subdomains containing DGAT1 or ER export sites. We show also using split-ubiquitin membrane yeast-two hybrid assays that GPAT8 interacts with itself, GPAT9 and DGAT2, but not with DGAT1. Furthermore, mutational analysis of GPAT8 indicated that the protein's first transmembrane domain (TMD), a sequence that contains a leucine zipper-like motif known to be involved in the interactions and assembly of various other membrane-bound proteins, mediates ER subdomain localization. Taken together, these results suggest that the regulation and organization of ER subdomains involved in lipid biosynthesis in plant cells is mediated by higher-ordered, TMD-dependent protein homo- and hetero-oligomeric interactions.